Specificity of transposition of Tn7 in Vibrio parahaemolyticus.

Tn7 was found to transpose at a high frequency from the plasmid, RP4::Tn7, to the chromosome of Vibrio parahaemolyticus. Seven isolates carrying Tn7 insertions were derived from three wild-type strains isolated from geographically distinct areas, and HindIII and BstEII DNA digests of these strains were probed with a ColE1::Tn7 biotinylated probe. The results indicated that V. parahaemolyticus is similar to several other species which have been studied in having a highly preferred site of insertion of Tn7 in the chromosome.

Bacterial detoxification of diisopropyl fluorophosphate.

The ability of 18 gram-negative bacterial isolates to detoxify diisopropyl fluorophosphate, a structural analog of the agents soman and sarin, was investigated. Detoxification by both frozen cell sonicates and acetone powders was assayed by two methods, i.e., the hydrolytic release of fluoride, measured by a fluoride-specific ion electrode, and the disappearance of acetylcholinesterase inhibition in vitro. Frozen cell sonicates for all strains exhibited some activity (F- ion release). In general, acetone powder preparations produced higher activity than frozen cell sonicates did, and the highest activities were exhibited by strains with known parathion hydrolase activity. Two ranges in activity were observed, low level, ranging from 0.1 to 7.0 mumol/min per g of protein, and high level, detected only in parathion hydrolase-producing strains, from 47 to greater than 300 mumol/min per g of protein. Results indicate that parathion hydrolase was nonspecific in phosphoesterase activity. Also, it was an effective detoxicant at low concentrations and near-neutral pH.

Novel transmissible factors in a non-O1 Vibrio cholerae and a Vibrio sp.

Transmissible factors encoding production of lacunae (L factors) were demonstrated in a non-O1 Vibrio cholerae and a Vibrio sp. of recent environmental origin. Lacunae were produced in lawns of non-O1 V. cholerae indicator strains under the same assay conditions as those where lacunae were produced by the well characterized P fertility plasmid of V. cholerae O1 and the V fertility factor found in a non-cholera vibrio strain. The origin of the lacunae produced by strains harbouring the V and L factors was examined. No vibriocin or phage activity was found in culture supernates or in lacunae produced by the strains, suggesting that, as in the case of the P plasmid, the lacunae probably represent sites of active mating. Unlike the P plasmid, neither the Vn or L factor could be detected or isolated by conventional plasmid techniques.

Serology and plasmid carriage in Vibrio cholerae.

Vibrio cholerae strains of the 01 serovar, isolated from both clinical and environmental sources, had a much lower frequency of plasmid carriage (2/112 = 2%) than clinical and environmental non-01 serovar isolates (46/187 = 25%). The cryptic plasmids found in non-01 strains were all of low molecular weight and were shown by hybridization analysis to consist of two unrelated subgroups. Each subgroup was observed to be present in strains isolated from both clinical and environmental sources. One 01 serovar (ATCC 14033) carried a small cryptic plasmid, belonging to one of these plasmid groups, while a second (25728) contained a high molecular weight multiple antibiotic resistance plasmid which did not hybridize to either plasmid subgroup.

Isolation and characterization of small plasmids in strains of Vibrio parahaemolyticus.

PIP: Vibrio parahaemolyticus strains isolated from patients and foods incriminated in 4 incidents of gastroenteritis, as well as a strain from a healty carrier were analyzed for plasmids. Large plasmids were found in a Kanagawa phenomenon positive (KP+) patient isolate from Louisiana of 04:K8 serotype but not in KP+ patient isolates of 2 other serotypes from the same outbreak. Small plasmids in the molecular weight range of 5 to 9 Mdal were found in 4 other strains isolated in Bangkok, Africa and the United States. The small plasmids did not share a high degree of molecular relatedness as indicated by dissimilarity in restriction enzyme patterns. However, DNA probe hybridization of restriction digests revealed some partial DNA sequence homologies. The presence of plasmids in the 11 strains examined did not correlate with the Kanagawa phenomenon, antibiotic resistance or production urease.^ieng

Isolation of drug-resistant Aeromonas hydrophila from aquatic environments.

Antibiotic-resistant strains of Aeromonas hydrophila have been isolated from the natural environment in the Chesapeake Bay and areas surrounding Dacca and the Matlab region of Bangladesh. The Bangladesh strains carried resistance to chloramphenicol, streptomycin, and tetracycline, and 57% of them had a multiple streptomycin-tetracycline resistance phenotype correlated with the presence of a large plasmid. The Chesapeake Bay strains were resistant to polymyxin B ane tetracycline, but showed neither multiple resistance nor R-factor carriage. Twenty-five percent of the environmental strains were toxigenic in a Y-1 adrenal cell assay. Toxigenicity showed no positive correlation with drug resistance or with plasmid carriage. Environmental areas of heavy human impact appear to be associated with a higher incidence of antibiotic-resistant strains of aeromonads.

Isolation and characterization of spontaneously arising auxotrophic and Kanagawa phenomenon-negative mutants of Vibrio parahaemolyticus.

As a first step toward developing a system of genetic exchange between Vibrio parahaemolyticus strains, spontaneously arising auxotrophic and Kanagawa phenomenon-negative (KP-) mutants were isolated and characterized. Auxotrophic mutants were selected by nalidixic acid enrichment of parental cultures. Some Cys- and Arg- mutants of a KP+ strain were found to be KP-. Reversion to prototrophy by these strains was not accompanied by a return to the parental KP+ phenotype. Additionally, two prototrophic KP- mutants were isolated. No detectable levels of vibriolysin were found in supernatant extracts of KP- mutants by slide gel immunodiffusion analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, or assay for lethal activity in mice. All Cys-, Arg-, and Pur- mutants tested reverted to a different auxotrophy (phenotypic interconversion) as well as to prototrophy. The possible role of insertion sequence-like elements in vibriolysin production and phenotypic interconversion is discussed.

his-Linked hydrogen sulfide locus of Salmonella typhimurium and its expression in Escherichia coli.

A his-linked H2S locus of Salmonella typhimurium has been further defined by direct isolation of H2S mutants. Expression of this locus in Escherichia coli has been demonstrated.

Effects of microbial activity on aquatic pollutants.

Bacteria and fungi present in estuarine and marine water and sediment accomplish significant degradation of crude oil, refined oils, polychlorinated biphenyls, and organomercurials, with the rate and extent of degradation varying with species, geographic source, temperature, and other biologic and environmental parameters. Our biodegradation studies have been extended to determine if physical weathering and/or microbial degradation of oil by microorganisms present in Chesapeake Bay water and sediment produces potentially carcinogenic substances. Water and sediment from an area in Chesapeake Bay that receives heavy input of oil and from a relatively nonpolluted site have been assayed for mutagenic ability by use of the Ames method, which is a bacterial assay and is highly sensitive. Preliminary findings indicate the presence of mutagenic substances in samples collected from the polluted site. Extracts of oil subjected to microbial degradation under controlled laboratory conditions did not yield detectable mutagenic activity. In situ studies are in progress.

Genetic characterization of a phi80 transducing bacteriophage carrying the histidine operon of Salmonella typhimurium.

A phi 80 transducing phage, phi 80imm lambdadhis, carrying the Salmonella his-gnd region, was characterized by immunity studies, tonB deletion analysis, and marker rescue analysis. Phi 80imm lambdadhis retains the phage immunity region of the phi 80-lambda hybrid phage from which it was derived. Bacterial genes replace most late phage genes. Deletion analysis shows the prophage gene order to be immlambda-his-gnd and indicates the orientation of the his operon to be hisOGDCBHAFIE-gnd. The structure of phi 80imm lambdadhis is remarkably similar to two independently isolated phi 80 phages that carry the his-gnd region of Escherichia coli and that, like phi80imm lambdahis, were derived by directed gene transposition to the tonB locus. A derivative of phi 80imm lambdadhis that is phi 80 immune is also reported.

His-linked hydrogen sulfide locus in Salmonella typhimurium.

Investigation of strains of Salmonella typhimurium having extended his deletion mutations indicates that a genetic site (or sites) affecting H(2)S production is located in the region of the chromosome adjacent to the operator end of the his operon. This site is co-transducible with the his genes. Experimental data indicate that the site is also present on an F'his factor derived from S. typhimurium, FS401. Evidence is presented for the existence of another site affecting H(2)S production which is not linked to his.

Nalidixic acid for enrichment of auxotrophs in cultures of Salmonella typhimurium.

Nalidixic acid kills only growing cells of Salmonella typhimurium and can be used to enrich for auxotrophs in populations where prototrophs predominate.

Derivation of an F-merogenote and a phi-80 high-frequency transducing phage carrying the histidine operon os Salmonella.

An F' factor, FS400, carrying the his operon, the gnd gene, and the rfb gene cluster of Salmonella typhimurium was isolated. FS400 was introduced into an Escherichia coli strain having a lengthy deletion of the his gene region. From this strain, Hfr derivatives were isolated which had the F' factor integrated in the tonB locus near the attachment site of phi80. One of the Hfr strains was lysogenized with a heat-inducible, h mutant of phi80, and from this strain a high-frequency transducing phage carrying the his genes and the gnd gene of Salmonella was isolated.

Actinomycin resistance and actinomycin excretion in a mutant of Escherichia coli.

A mutant of Escherichia coli resembles its parent in taking up actinomycin after treatment with ethylenediaminetetraacetic acid but differs in that it survives this uptake and excretes actinomycin at an increased rate.